Biosynthesis of waxy esters



Feb, 9, 1965 J. B. DAVIS BIOSYNTHESIS OF WAXY ESTERS Filed Oct. 31. 1961 INVENTOR: By John BDaz/Ls ATTORNEY 3,169,099 urosYNrnnsrs on waxy EdTERS John E. Davis, Deltas, Tex assignor to Socony Mebii @ii Qornpany, inc, a corporation of New York Fiied Oct. 31, 1961, Ser. No. 149,531

Claims. (Ci. 195-3) This invention relates to the utilization of'hydrocarbons by micro-organisms. More particularly, it relates to the conversion of hydrocarbons to Waxes as intercellular products, among other products, by microbes.

The production of waxes as extra-cellular products by certain bacteria has been reported, but a practical conversion has hithertofore not been developed. One difficulty stems from the fact that bacteria that utilize hydrocarbons more readily use other sources of energy such as fats,'carbohydrates and proteins. Another problem has been the relatively slow growth rate of microorganisms. Since the conversion of the inexpensive hydrocarbons to oxygenated and other products is desired and since microbes effect oxidations and conversions under inexpensive temperature (about 75 to above 115 F.) and pressure (atmospheric) conditions, efiicient routes to products from hydrocarbons using microbes has been actively sought in recent years.

One such development is described and claimed in copending application Serial No. 149,112 and now US. Patent No. 3,131,131. In that advance, beta-carotene is produced by nocardia along with other products which include waxes. While carotenoids are produced in general by yellow-orange pigmented nocardia from hydrocarbons in general, the production of aliphatic waxes by the micro-organism is limited to certain hydrocarbons.

Accordingly, an object of this invention is the provision of a system in whichmicrobes'are cultivated on hydrocarbons with the production of waxes. Another purpose is the conversion of long chain hydrocarbons to esters from long chain aliphatic carboxylic acids and long chain aliphatic alcohols. A still further aim is preparation of such aliphatic esters in good yields by the action of microbes growing under conditions that permit their rapid growth. These and other objectives, will appear hereinafter.

The objects of this invention are accomplished by placing a microbe of the genus Nocardia in an oxidator and intimately contacting the microbial cells with oxygen, an aqueous, mineral nutrient anda hydrocarbon, allowing the cells to grow. on the hydrocarbon and to multiply and then concentrating the resultant reaction mixture to produce a concentrate of wax. The reactants, including the metabolizing cells, are vigorously agitated to effect the desired contact. The hydrocarbons are water insoluble, as are the cells but contact must be made between the cells and the hydrocarbon and between the cells and the aqueous nutrient solution. By violent agitation the hydrocarbon is broken down into small globules. By observation with a microscope one can readily see the strong afiinity for the globules. Rapid cellular growth and division result. On the other hand if the'hydrocarbon is added in bulk or under conditions in which it floods the cellular material, the cells become-embedded in the hydrocarbon and have no access to the needed minerals; growth is poor and wax yields are low. The nocardia are used in preference to other micro-organisms because they multiply at a much faster rate. For example, the generation time Qfnocardia is about 30 minutes whereas for mycobacter ia the time is in the order of 24 hours. Not only do the Nocardia grow well on the hydrocarbons used in this invention but they grow much faster than do other microsorganisms. Etlicient production of waxes results.

iiihihh fih Patented Feb. 9, 1065 From the above it can readily be appreciated that the processes involved are chemical processes or manners of new manufacture requiring an operator who maintains appropriate conditions and drives the micro-organisms to the desired results. The operator separates the resultant mixtures or the desired products for further utilization as desired.

This invention will be further understood by reference to the description, the figures, and the examples below, given for illustrative purposes and not to be taken as limitative, the figures being as follows:

FIGURE 1 is a perspective of apparatus used in this invention;

FIGURE 2 shows the impeller used in this invention; and

FIGURE 3 shows the sparger through which fluid materials maybe added to the media to be oxidized.

As can be seen from FIGURE 1, the oxidator l comprises a vessel 2 having a lid 3,, the two being held together by flanges through bolting members 5. Positioned inside the vessel 2 is a stainless steel fritted sparger 6 which comprises two circular rings '7 connected by uprights 8 which coact with the impeller 9 to impart violent agitation and a shearing action to the contents. The impeller is motor driven at M through shaft 10 positioned to rotate in a graphite impregnated bronze bearing mounted in a neoprene plug at 11 in the top 3. At the bottom of the sparger is a perforated disk 12. which has tube 13 leading to the perforations in sealed arrangement to deliver materials through the perforations and into the charge, the inlet being at 14. Pressure Within the vessel may be kept at atmospheric pressure, the feeding of the gaseous materials being commensurate with their utilization by the microbes. If desired, excess materials may he passed through outlet 15 to be recirculated, sent to another oxidator or collected, using conventional pumping means when needed. Thus, the processes of this invention encompass running a plurality of oxidators in a tanker in a continuous operation. Since the processes of this invention involve aerobic conditions, normally oxygen or air will be supplied along with food materials. Therefore, outlet 15 is usually a part of the apparatus, and in many instances this can be simply an opening plugged with cotton or other porous material.

As is well known, microbes can be grown on cultures containing mineral salts as a source of essential food materials. These media may be varied widely in their components, both as to identity and to concentration. in this invention the following culture medium has been used frequently, the amount in parentheses being the number of grams used per liter of distilled water: am-

. monium sulfate (1.0) disodium hydrogen phosphate (0.3), potassium dihydrogen phosphate (0.2), magnesium sulfate septahydrate (0.2), sodium carbonate (0.1), calcium chloride (0.01), ferrous sulphate septahydrate and manganous sulfate (0.002). In order to have on hand cultures of the said Nocardia, it was found convenient to growthe said Nocardia on 0.4% n-octadecane and 250 ml. bottles incubated on a rotary shaker at 30 C.

In the following examples, parts and percentages are .by weight unless otherwise noted.

EXAM'PLE I Using apparatus such as shown in FIGURE 1, a small amount of Nocardia salmonicolor, strain 107-332, was added to a liter of above aqueous salt solution and then the hydrocarbons listed in Table 1 below were added to the cells and oxygen was fed through the sparger and while violently agitating the mixture. The biosynthetic salt mixture and the cells were separated by centrifugaan J. The isolated cells were extracted and tested for The results follow:

T able 1 WAXES FOUND IN LIPID FRACTION tion. lipid and wax contents.

From the above it can be seen that wax is not obtained from all hydrocarbons. The C wax is C in the alkyl and C in the acyl part of the ester. The C is C in both moieties; the C is C and C respectively, while the C is C in both. Thus, the waxes include cetyl myristate, cetyl palmitate, stearyl palmitate and stearyl stearate.

in an experiment in which 10 grams of n-octadecane was consumed, grams of cellular lipid resulted. Of this about 80% was stearyl stearate and was stearyl palmitate.

When Nocara'ia corailinr'z, strain M.O., is used instead of a Nocarzlia salmoizicolor, similar results are obtained.

, Short chain and odd-numbered allranes are inactive to yield waxes.

*XAMPLE ii To a literof the aqueous mineral solution described above was added about ml. of a nocardia culture, and the resultant medium was air agitated as described above. n-Hexadecane was then added to a 'l% concentration and more was added as the hydrocarbon was used up, the experiment being stopped when the cells constituted about 20 grams/ liter.

The cells were then separated by centrifugation and dried. The lipid fraction was isolated by extracting the dried cells with a fat solvent such as ethyl ether. This fraction contained the wax along with other lipid components such as glycerides. Separation of the wax from the other lipid components Waseil'ected by adding the ethyl ether solution of the nocardia cellular lipid to an equal volumeof acetone. The WZIXBS precipitated and were recovered by filtration.

The wax fraction represented 40% of the cellular lipid, and the triglycerides constituted Lipid yields as high as 7.0% Were obtained in other experiments.

Results similar to the above were obtained with n-octa- EXAMPLE III decane.

8 g. of n-octaclecane (0.4%) was added and the stirrers were rotated at 1750 rpm. Hydrocarbon was added as it approached deletion in the system. After 60 hrs. 8.3

' g. of nocardial cells was obtained from 11.8 g. of the hydrocarbon yield). In a similar experiment an yield was obtained; and in still another experiment 48.8 g. of cells Was obtained from 57.0 g. of the hydrocarbon, this corresponding to an yield.

There is also produced, but as an extracellular product, a polymeric material being capsular slime and a polysaccharide. In following the total conversion, it was found that lb. of the n-alkane or" this example yielded 0.7 lb. of microbial cells and 0.25 lb. of the said polymer. This is a conversion.

The polymer is carbohydrate in nature, is soluble in Water and is of a high molecular weight, for some of it is centrifuged out of apparent solution at 35,000xg.

or of the microbes.

Upon hydrolysis the solution gives a weak test for reducing sugar, and the polymer contains no nitrogen. When other long chain hydrocarbons, such as n-dodecane, are substituted for the n-octadecane above there is formed corresponding long chain aliphatic esters from the relative long chain alcohols and acids resulting from oxidation of the hydrocarbon by the nocardia.

EXAMPLE IV In conducting experiments such as the above it was noted that the nitrogen source was depleted limiting growth and also that the pH dropped requiring buffering or neutralization with alkali. Accordingly, runs were made varying the source of nitrogen and the amount of the nitrogenous compound added. The results'are given Table II below showing the nitrogen source, the percent used based on the liquid nutrient, the amount of hydrocarbon used, and yield of dried cells, the amount of the lipid fraction obtained and the final pH.

, Table II EFFECT OF NITROGEN SOURCES ON NOCARDIAL CELL YIELDS AND LIPID FORMATION From the above it can be seen that the higher the amount of nitrogen the lower the yield of lipid fractions. The result with urea is noteworthy in that a lower lipid ield is obtained even though the pl-l is not lowered.

From the results it was concluded that it is preferred to use nitrogen compounds in amounts no greater than 0.1% based on the weight of the aqueous nutrient that is to receive the initialsmallinoculate of the micro-organism. While with proteins such as asparagine no serious effect is noted, the cell lipid fraction being only mildly reduced, there appears to be no advantage in using the higher amounts of organic nitrogen sourcesj Normally, the various steps of the processes of this invention are effected at room temperatures. However, operations in the cold are possible and certain steps have been effected successfully at temperatures around 115 F.

The feed stream may be warmed before passing it to theit is also to be understood that the processes can be operated under pressure if desired and that a small amount of cells-or a large amount of cells may be initially present. The process may be started immediately, no incubation period nor a certain amount of cells being required. for

One will adjust the amount of mineral substrate, the rate of hydrocarbon feed, the rate of productremoval, and the like in keeping with optimum growth. In the experiments described above air or vapor teed rates have been in the range of about to about 1000 ml./l./min., but slower and much greater feed rates can, of course, be used. Generally, the pH will be kept within the range of about 6.0 to about 8.0 with a pH of 7 .0 being preferred. Frequently, to prevent acidity and to aid growth rates phosphate salts are added. From the above, it can be seen that eflicient, smooth-running and readily controlled processes are afforded by this invention.

Oddly, the eiTect of oxygen levels is not a straight-line matter. Nocardia (Nocardia salmom'color, strain 107-332) was grown for 24 hours in air on n-octadecane and then grown an additional 24 hours at varied oxygen concen tations: 20%, and 2%. The best yields of lipid fractions was 73% obtained a being 56% at the 20% level and 21% at the 2% level.

Another condition having an effect on microbial growth is the concentration of hydrocarbon or the manner in which the hydrocarbon is fed. For example, the addition of 24 gams of n-octadecane to 2 liters of mineral salts medium containing Nocardia salmonicolor, strain 107- 332, led to no growth. It appears from microscope studies in this experiment that the cells became embedded in the hydrocarbon and got isolated from the aqueousmediurn. In this experiment the microbes present amounted to 1.2 grams so that the ratio of hydrocarbon to cells was 20 to l on a weight basis. Under identical conditions save for a step-wise addition of the hydrocarbon, proper growth was attained. At the start 8 grams of the n-octadecane was added, followed by additional 8 gram portions at 24, 42 and 48 hours. The cells were growing well when the experiment was terminated. A total of 13.8 grams of cells (dry wt.) was obtained, 18 grams of the hydrocarbon having been utilized. In this experiment the initial ratio of hydrocarbon to microbial cellswas 7 to 1 on a weight basis. While it is possible to tolerate high concentrations by violent agitation, it is preferred to add the hydrocarbons at rates commensurate with their utilization by the microbes.

The hydrocarbons used in this invention are long chain, even-numbered n-alkanes. Short chain n-alkanes, such as propane, butane or n-hexane, do not yield waxes even though nocardia utilize such hydrocarbons for growth. The odd-numbered hydrocarbons, such as n-tridecane and n-nonadecane do not yield waxes even through the nocardia grow very well on these hydrocarbons. Other long chain n-alkanes which may be used in the process of this invention include n-hexadecane, n-tetradecane, n-octadecane, n-eicosane, n-dodecane, among others.

The nocardia used in theprocess of this invention are any of the genus Nocardia. Generally, a species is isolated by enrichment procedures, as, for example, by ethane enrichment using soil plating procedures. Those cultures growing well in ethane are then kept available growing on hydrocarbons that are liquids or solids, such as noctadecane. Nocardza salmonicolor, strain 107-332, and N ocardia corallina, strain M .O., are two such nocardia that may be used in the production of waxes. The various the 10% level, the yield nocardia are rapid growers on hydrocarbons, affording more chemical conversion per unit of time than other known hydrocarbon oxidizers.

A wide variety of methods may be used to concentrate or to isolate the waxes produced in this invention. Generally, the cells are recovered by filtration or centrifugation. The cells, dried if desired, are then extracted with a solvent for lipids such as ethyl ether, chloroform, hexane, benzene, chloroform-methanol (2:1) and the like. Extraction in a ball mill can be effected also, the milling bringing about a pulverization of the cellsor destruction of protective cell walls that facilitates extraction. Any unchanged alkane may be recovered in the extractive step or by chromatographic separation on an activated silica gel column, for example, or by. fractionation in distillation procedures. Generally, the wax is separated from glycerides on a solubility basis, the waxes being insoluble in cold acetone which dissolves the glycerides. Redissolving and precipitation again can be used to purify the waxes.

By the processes of this invention the rapid utilization of long chain n-alkanes is effected with their conversion to waxes, among other products, by nocardia. Very high conversions of the alkanes, up to can be effected. Further, the products can be readily concentrated, isolated and purified. The retention of the waxes within the cells is an advantage for with the intercellular production of chemicals, desired products, such as the waxes, are conveniently localized for immediate processing either in batch-wise or in continuous productions.

While the invention has been disclosed herein in connection with certain embodiments and certain structural details, it is clear that changes, modifications or equivalents can be used by those skilled in the art; accordingly, such changes within the principles of this invention are intended to be included within the scope of the claims below.

What is claimed:

1. A process for the production of a waxy ester from a hydrocarbon by a micro-organism which process comprises aerobically subjecting a long chain, even-numbered n-alkane to the metabolic action of nocardia in the presence of an aqueous mineral nutrient containing a source of nitrogen for said nocardia, which nutrient contains no more than about 0.3% by weight, based on the weight of said nutrient, of said nitrogen compounds; allowing the cells to grow and multiply and to convert the said hydrocarbon into products containing waxy ester; and concentrating the said waxy product.

2. A process in accordance with claim 1 in which said hydrocarbon is n-hexadecane.

3. A process in accordance with claim 1 in which said hydrocarbon is n-octadecane.

4. A process in accordance with claim 1 in which said nocardia is Nocardia salmonicolor, strain M.O. 107-332.

5. A process for the production of a waxy ester from a hydrocarbon by a micro-organism which process comprises placing a nocardia culture in a vessel; passing oxygen to the microbial cells in said culture while the said cells are in the presence of and in contact with a long chain, even-numbered n-alkane and with an approximately neutral, aqueous nutrient containing nitrogen compounds as a source of nitrogen for said nocardia, which nutrient contains no more than about 0.3% by weight, based on the weight of said nutrient, of said nitrogen compounds; intimately contacting the said cells with the said oxygen, hydrocarbon and nutrient; allowing the cells to grow and multiply and to convert the said hydrocarbon to a product that contains a waxy ester; and concentrating the said waxy product.

6. A process in accordance with claim 5 in which the reaction medium comprising the said cells, oxygen, hydrocarbon and nutrient is kept at a pH of about 6.0 to about 8.0.

7. A process in accordance with claim 5 in which the nitrogen con-tent of said nutrient is not more than about 0.1% by weight of the aqueous nutrient.

8. A process in accordance with claim 5 in which said hydrocarbon is n-hexadecane.

9. A process in accordance with claim 5 in which said hydrocarbon is n-octadecane. 1

10. A process in accordance with claim 1 in which the said waxy ester comprises cetyl myristate.

11. A process in accordance with claim 1 in which the said waxy ester comprises cetyl palmitate.

12. A process in accordance with claim 1 in which the said waxy ester comprises stearyl palmitate.

13. A process in accordance with claim 1 in which the said waxy ester comprises stearyl stearate.

14. A process for the production of a waxy ester from a hydrocarbon by a microorganism which process comprises aerobically subjecting a long-chain, even-numbered n-alkane to the metabolic action of nocardia in an aqueous nutrient which contains no more than about 0.1% by weight, based on the weight of said nutrient, of nitrogen compounds available to said nocardia as food; intimately contacting the said nocardia with a source of oxygen for said aerobic metabolism; allowing the cells to grow and 3,169,099 'I'" i3 multiply and to convert the said hydrocarbon into products OTHER REFERENCES ggg gg gf g ga t fig gi g i mcentmmlg Beerstecher, Jr.: Petroleum Microbiology, Elsevier 15. A process in accordance with claim 14 in which Press Houston f air is the source of oxygen and the said aqueous nutrient 5 webleyft Journal Of Blochemlstry, 3714, is kept at a pH of about 6.0 to about 8.0. Q 5 1 3 References Cited by the Examine A; LOUIS MONACELL, Primary Examiner.

UNITED STATES PATENTS ABRAHAM H WINKELSTEIN E- 2,338,207 1/44 'Shappirio 1 3 10 v m- 2,742,398 4/56 Z0 Bell. 

1. A PROCESS FOR THE PRODUCTION OF A WAXY ESTER FROM A HYDROCARBON BY A MICRO-ORGANISM WHICH PORCESS COMPRISES AEROBICALLY SUBJECTING A LONG CHAIN, EVEN-NUMBERED N-ALKANE TO THE METABOLIC ACTION OF NOCARDIA IN THE PRESENCE OF AN AQUEOUS MINIERAL NUTRIENT CONTAINING A SOURCE OF NITROGEN FOR SAID NOCARDIA, WHICH NUTRIENT CONTAINS NO MORE THAN ABOUT 0.3% BY WEIGHT, BASED ON THE WEIGHT OF SAID NUTRIENT, OF SAID NITROGEN COMPOUNDS; ALLOWING THE CELLS TO GROW AND MULTIPLY AND TO CONVERT THE SAID HYDROCARBON INTO PRODUCTS CONTAINING WAXY ESTER; AND CONCENTRATING THE SAID WAXY PRODUCT. 